Biomedical subjects

Isao Morishima

Publications and source records attributed to Isao Morishima.

At least 37 records · Page 2Linked to original sources

PubMed2004

NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle.

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N(2)O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe(B). The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe(B)-NO and the penta-coordinated ferrous heme b(3)-NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N(2)O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.

Electron Spin Resonance Spectroscopy↗

PubMed2004

Activation mechanisms of transcriptional regulator CooA revealed by small-angle X-ray scattering.

CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration Rg and the second virial coefficient (A2) were 25.3(+/-0.5)A and -0.39(+/-0.25) x 10(-4)ml mol g(-2), respectively. CO binding caused a slight increase in Rg (by 0.5A) and a marked decrease in A2 (by 5.09 x 10(-4)ml mol g(-2)). The observed decrease in A2 points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of Rg rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental Rg and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8 degrees around the axis of 2-fold symmetry.

Amino Acids↗

PubMed2004

L358P mutation on cytochrome P450cam simulates structural changes upon putidaredoxin binding: the structural changes trigger electron transfer to oxy-P450cam from electron donors.

To investigate the functional and structural characterization of a crucial cytochrome P450cam (P450cam)-putidaredoxin (Pdx) complex, we utilized a mutant whose spectroscopic property corresponds to the properties of the wild type P450cam in the presence of Pdx. The 1H NMR spectrum of the carbonmonoxy adduct of the mutant, the Leu-358 --> Pro mutant (L358P), in the absence of Pdx showed that the ring current-shifted signals arising from d-camphor were upfield-shifted and observed as resolved signals, which are typical for the wild type enzyme in the presence of Pdx. Signals from the beta-proton of the axial cysteine and the gamma-methyl group of Thr-252 were also shifted upfield and down-field, respectively, in the L358P mutant as observed for Pdx-bound wild type P450cam. The close similarity in the NMR spectra suggests that the heme environment of the L358P mutant mimics that of the Pdx-bound enzyme. The functional analysis of the L358P mutant has revealed that the oxygen adduct of the L358P mutant can promote the oxygenation reaction for d-camphor with nonphysiological electron donors such as dithionite and ascorbic acid, showing that oxygenated L358P is "activated" to receive electron from the donor. Based on the structural and functional characterization of the L358P mutant, we conclude that the Pdx-induced structural changes in P450cam would facilitate the electron transfer from the electron donor, and the Pdx binding to P450cam would be a trigger for the electron transfer to oxygenated P450cam.

Ascorbic Acid↗

PubMed2004

Crystal structure of the cytochrome p450cam mutant that exhibits the same spectral perturbations induced by putidaredoxin binding.

The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata-Ariga, Y., Egawa, T., Suematsu, M., Park, S.-Y., Adachi, S., Shiro, Y., and Ishimura, Y. (2003) Biochemistry 42, 14507-14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying 1H NMR paper (Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. (2004) J. Biol. Chem. 279, 42836-42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous (Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with non-physiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.

Aspartic Acid↗

PubMed2004

Human neuroglobin interacts with flotillin-1, a lipid raft microdomain-associated protein.

Neuroglobin (Ngb) is a newly discovered vertebrate globin that is expressed in the brain and that can reversibly bind oxygen. It has been reported that Ngb levels increase in neurons in response to oxygen deprivation, and that it protects neurons from hypoxia. However, the mechanism of this neuroprotection remains unclear. Recently, we found that oxidized human Ngb bound to the alpha-subunits of heterotrimeric G proteins (Galpha) and acted as a guanine nucleotide dissociation inhibitor for Galpha. To identify other Ngb-binding proteins, we herein screened a human brain cDNA library by using a yeast two-hybrid system. Among the plasmids isolated from positive clones, one contained an insert with 100% sequence identity to human flotillin-1. The interaction of Ngb with flotillin-1 was confirmed by glutathione S-transferase pull-down experiments. Since Galpha exists within lipid rafts critical for signal transduction and flotillin-1 recruits signaling proteins to lipid rafts, flotillin-1 might recruit Ngb to lipid rafts as a means of preventing neuronal death.

Amino Acid Sequence↗

PubMed2004

Association of human neuroglobin with cystatin C, a cysteine proteinase inhibitor.

Neuroglobin (Ngb) is a newly discovered globin that is expressed in vertebrate brain. It has been reported that Ngb levels increase in neurons in response to oxygen deprivation, and that Ngb protects neurons from hypoxia. However, the mechanism of this neuroprotection remains unclear. In the present study, we identified human cystatin C, a cysteine proteinase inhibitor, as an Ngb-binding protein by using a yeast two-hybrid system. Surface plasmon resonance experiments verified that Ngb binds to cystatin C dimers, not to the monomers. Because both intracellular cystatin C and the amyloidogenic variant of cystatin C form dimers, Ngb may modulate the intracellular transport (or secretion) of cystatin C to protect against neuronal death under conditions of oxidative stress and/or it may have a role in the development of neurodegenerative diseases.

Amino Acid Sequence↗

PubMed2004

Dioxygen reduction by bo-type quinol oxidase from Escherichia coli studied by submillisecond-resolved freeze-quench EPR spectroscopy.

The mechanism of the dioxygen (O(2)) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy. The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O(2)-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms. The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms. The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after 1 ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o. In contrast, the enzyme without the bound ubiquinone-8 (Delta UbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the F(I) intermediate possessing ferryl-oxo heme o, cupric Cu(B), and ferric heme b is converted to the F(II) intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the F(II) intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center. Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and Cu(B) and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions.

Binding Sites↗

PubMed2004

Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.

The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.

Animals↗

PubMed2004

The role of the DRY motif of human MC4R for receptor activation.

We constructed several mutant human MC4R cDNAs by site directed mutagenesis and expressed these receptors in COS-1 cells. The conserved DRY motif among GPCRs was mutated to generate eight mutants. While no MC4R ligand binding was detected in any of the mutants, one mutant, D146A, resulted in higher cAMP production in cells than the wild-type receptor without ligand stimulation.

Amino Acid Motifs↗

PubMed2004

Regulation of CRF, POMC and MC4R gene expression after electrical foot shock stress in the rat amygdala and hypothalamus.

We investigated the effects of electrical foot shock stress on the melanocortin signaling cascade and the hypothalamus-pituitary-adrenal (HPA) system by observing levels of mRNA expression of corticotropin releasing factor (CRF), pro-opiomelanocortin (POMC), and melanocortin receptor subtype 4 (MC4R) in the rat amygdala and hypothalamus. When rats were exposed to electrical shock for 0.5 hr or 1 hr, plasma ACTH and corticosterone concentrations increased, indicating stress. The rats were then sacrificed to obtain RNA preparations from the brain tissue. In the amygdala, the expression of MC4R and POMC mRNA as well as CRF mRNA was significantly increased by electrical foot shock stress. In the hypothalamus, MC4R and POMC mRNA increased, but CRF mRNA remained unchanged. The duration of increased gene expression of MC4R and POMC in the amygdala was more sustained than in the hypothalamus. These results have provided the first evidence that exposure to stress increases expression of the MC4R system in the amygdala and hypothalamus.

Amygdala↗

PubMed2003

Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2alpha kinase.

The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His78 and His123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His123 appears to act as the "distal" heme ligand.

Amino Acid Sequence↗

PubMed2003

Kinetic and spectroscopic characterization of a hydroperoxy compound in the reaction of native myoglobin with hydrogen peroxide.

The reaction of metmyoglobin with H2O2 was investigated in a pH range between 8.5 and 6.0 with the aid of stopped flow-rapid scan and rapid freezing-EPR techniques. Singular value decomposition analyses of the stopped flow data at pH 8.5 revealed that a spectral species previously unknown accumulated during the reaction and exhibited a Soret absorption maximum at >/=423 nm. In the EPR experiments, the new species exhibited a set of g values at 2.32, 2.19, and 1.94, indicating that the species was assignable to a ferric hydroperoxy (Fe(III)[O-O-H]-) compound. In contrast, the hydroperoxy compound scarcely accumulated in the reaction at pH 6.0, and the dominant intermediate species accumulated was compound I, which was derived from the oxygen-oxygen bond cleavage of the hydroperoxy compound. The accumulated amount of the hydroperoxy compound relative to compound I showed a pH dependence with an apparent pKa (pKaapp) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKaapp paralleled that in pKa of the acid-alkaline transition (pKaAB) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O2 activation by metmyoglobin is promoted at the acidic condition due to the imidazolium form of the distal histidine, and we further propose that the controlled protonation state of the distal histidine is important for the facile O-O bond cleavage in heme peroxidases.

Animals↗

PubMed2003

Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine nucleotide dissociation inhibitor.

Neuroglobin (Ngb) is a newly discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. It has been reported that Ngb expression levels increase in response to oxygen deprivation and that it protects neurons from hypoxia in vitro and in vivo. However, the mechanism of this neuroprotection remains unclear. In the present study, we tried to clarify the neuroprotective role of Ngb under oxidative stress in vitro. By surface plasmon resonance, we found that ferric Ngb, which is generated spontaneously as a result of the rapid autoxidation, binds exclusively to the GDP-bound form of the alpha subunit of heterotrimeric G protein (Galphai). In GDP dissociation assays or guanosine 5'-O-(3-thio)triphosphate binding assays, ferric Ngb behaved as a guanine nucleotide dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP. The interaction of GDP-bound Galphai with ferric Ngb will liberate Gbetagamma, leading to protection against neuronal death. In contrast, ferrous ligand-bound Ngb under normoxia did not have GDI activities. Taken together, we propose that human Ngb may be a novel oxidative stress-responsive sensor for signal transduction in the brain.

Amino Acid Sequence↗

PubMed2003

NMR study on the structural changes of cytochrome P450cam upon the complex formation with putidaredoxin. Functional significance of the putidaredoxin-induced structural changes.

We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the beta-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 A. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo- and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15-0.7 A. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the gamma-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by approximately 0.25 A. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction.

Bacterial Proteins↗

PubMed2003

cDNA cloning and expression of bacteria-induced Hdd11 gene from eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding Hdd11 protein, a bacteria-induced protein of unknown function, was isolated from fat body of immunized Samia cynthia ricini larvae based on suppression subtractive hybridization. The cDNA encodes a 167 amino acid residue open reading frame with an 18 residue predicted signal peptide. The deduced amino acid sequence showed 54% and 55% identity with Hdd11 proteins from Hyphantria cunea and Manduca sexta, respectively. Expression of the gene was undetectable in naive larvae when measured by Northern blot hybridization, but strongly induced in fat body after injecting bacterial cells or peptidoglycan into the larvae. The mRNA expression in the fat body was detected as early as 3 h post injection, reached peak levels at 12 h and continued for further 60 h at significant levels. The transcript was detected at very low levels in midgut, hemocytes and malpighian tubules.

Amino Acid Sequence↗

PubMed2003

Activation of hydrogen peroxide in horseradish peroxidase occurs within approximately 200 micro s observed by a new freeze-quench device.

To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with approximately 200 micro s of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H(2)O(2)). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at approximately 1 ms after the mixing of HRP and H(2)O(2) suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at approximately 200 micro s show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within approximately 200 micro s after mixing HRP and H(2)O(2). We conclude that the activation of H(2)O(2) in HRP at ambient temperature completes within approximately 200 micro s. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.

Cold Temperature↗

PubMed2003

Identification of the ubiquitin-protein ligase that recognizes oxidized IRP2.

The ubiquitin system is involved in several basic cellular functions. Ubiquitination is carried out by a cascade of three reactions catalysed by the E1, E2 and E3 enzymes. Among these, the E3 ubiquitin-protein ligases have a pivotal role in determining the specificity of the system by recognizing the target substrates through defined targeting motifs. Although RING finger proteins constitute an important family of E3 ligases, only a few post-transcriptional modifications, including phosphorylation, proline hydroxylation and glycosylation, are known to function as recognition signals for E3. Iron regulatory protein 2 (IRP2), a modulator of iron metabolism, is regulated by iron-induced ubiquitination and degradation. Here we show that the RING finger protein HOIL-1 functions as an E3 ligase for oxidized IRP2, suggesting that oxidation is a specific recognition signal for ubiquitination. The oxidation of IRP2 is generated by haem, which binds to IRP2 in iron-rich cells, and by oxygen, indicating that the iron sensing of IRP2 depends on the synthesis and availability of haem.

Amino Acid Sequence↗

PubMed2002

Roles of the proximal hydrogen bonding network in cytochrome P450cam-catalyzed oxygenation.

Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated. A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L. The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P. The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively. Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I. J. Inorg. Biochem. 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause approximately 45 and approximately 35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam). The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482 approximately 483 cm(-)(1) compared with that of wild-type P450(cam) at 484 cm(-)(1). The Q360 mutants also showed the upshift by 4 approximately 5 cm(-)(1) of the Fe-NO stretching mode in the ferrous-NO form. These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the Q360 mutants and were in contrast to the increased pi-back-donation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-)(1). The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond. We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron. Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.

Camphor 5-Monooxygenase↗